RNAi-mediated knockdown of the poultry red mite cathepsin D-1 impacts haemoglobin digestion

Background The poultry red mite, Dermanyssus gallinae , is a haematophagous ectoparasite causing significant economic losses in the commercial egg-laying sector. Blood meal digestion by D. gallinae is required for nutrient acquisition, with acidic lysosomal proteinases such as cathepsin L and cathepsin D playing a critical role in haemoglobin digestion. This study investigated the role of a cathepsin D-like aspartyl proteinase, Dg-CatD-1, in the haemoglobin digestion cascade. Methods Haemoglobin processing was investigated by RNA interference (RNAi)-mediated silencing of Dg-CatD-1 and assessing the impact on haemoglobin digestion. RNAi-mediated knockdown of Dg-CatD-1 was achieved by feeding a target-specific double-stranded RNA (dsRNA) to D. gallinae in a blood meal. The minimum length and concentration of Dg-CatD-1 dsRNA for effective knockdown was determined. In addition, the effect of Dg-CatD-1 knockdown on mite digestive physiology, haemoglobin digestion, and egg-laying by adult female mites was assessed. Results Feeding Dg-CatD-1 dsRNAs via a blood meal to adult female D. gallinae mites resulted in a substantial knockdown of target gene expression. The minimum length and concentration of dsRNA required for effective Dg-CatD-1 knockdown were 25 base pairs (bp, at 200 ng/μl) (61% knockdown) and 25 ng/μl (at 485 bp) (42% knockdown), respectively. When Dg-CatD-1 dsRNA was delivered as a single feed it resulted in up to 91% reduction in Dg-CatD-1 expression, although no observable effect on blood digestion was observed. The phenotypic impact of Dg-CatD-1 knockdown was demonstrated following two consecutive rounds of Dg-CatD-1 dsRNA feeding where knockdown reduced the ability of mites to process and clear their blood meal relative to control non-specific dsRNA-fed mites. Conclusions This work highlights the importance of Dg-CatD-1 as an essential enzyme in the haemoglobin digestion pathway of D. gallinae . These findings open avenues for the development of targeted control strategies aimed at disrupting the digestive processes of D. gallinae . Furthermore, this research suggests that reductions in gene expression via RNAi do not always lead to corresponding decreases in protein levels or observable phenotypes. Repeated exposure to dsRNA may be necessary to reveal phenotypic effects of gene knockdown.